PU.1 and BCL11B sequentially cooperate with RUNX1 to anchor mSWI/SNF to poise the T cell effector landscape.

Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through nonspecific signaling intermediates is unclear. Here we examined the role of chromatin priming in specifying the functional outputs of effector T cells and found that most of the cis-regulatory landscape active in effector T cells was poised early in development before the expression of the T cell antigen receptor. We identified two principal mechanisms underpinning this poised landscape: the recruitment of the nucleosome remodeler mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) by the transcription factors RUNX1 and PU.1 to establish chromatin accessibility at T effector loci; and a 'relay' whereby the transcription factor BCL11B succeeded PU.1 to maintain occupancy of the chromatin remodeling complex mSWI/SNF together with RUNX1, after PU.1 silencing during lineage commitment. These mechanisms define modes by which T cells acquire the potential to elicit specialized effector functions early in their ontogeny and underscore the importance of integrating extrinsic cues to the developmentally specified intrinsic program.

AIRE in context: Leveraging chromatin plasticity to trigger ectopic gene expression.

The emergence of antigen receptor diversity in clonotypic lymphocytes drove the evolution of a novel gene, Aire, that enabled the adaptive immune system to discriminate foreign invaders from self-constituents. AIRE functions in the epithelial cells of the thymus to express genes highly restricted to alternative cell lineages. This somatic plasticity facilitates the selection of a balanced repertoire of T cells that protects the host from harmful self-reactive clones, yet maintains a wide range of affinities for virtually any foreign antigen. Here, we review the latest understanding of AIRE's molecular actions with a focus on its interplay with chromatin. We argue that AIRE is a multi-valent chromatin effector that acts late in the transcription cycle to modulate the activity of previously poised non-coding regulatory elements of tissue-specific genes. We postulate a role for chromatin instability-caused in part by ATP-dependent chromatin remodeling-that variably sets the scope of the accessible landscape on which AIRE can act. We highlight AIRE's intrinsic repressive function and its relevance in providing feedback control. We synthesize these recent advances into a putative model for the mechanistic modes by which AIRE triggers ectopic transcription for immune repertoire selection.

Genetic errors of immunity distinguish pediatric nonmalignant lymphoproliferative disorders.

BACKGROUND: Pediatric nonmalignant lymphoproliferative disorders (PLPDs) are clinically and genetically heterogeneous. Long-standing immune dysregulation and lymphoproliferation in children may be life-threatening, and a paucity of data exists to guide evaluation and treatment of children with PLPD. OBJECTIVE: The primary objective of this study was to ascertain the spectrum of genomic immunologic defects in PLPD. Secondary objectives included characterization of clinical outcomes and associations between genetic diagnoses and those outcomes. METHODS: PLPD was defined by persistent lymphadenopathy, lymph organ involvement, or lymphocytic infiltration for more than 3 months, with or without chronic or significant Epstein-Barr virus (EBV) infection. Fifty-one subjects from 47 different families with PLPD were analyzed using whole exome sequencing. RESULTS: Whole exome sequencing identified likely genetic errors of immunity in 51% to 62% of families (53% to 65% of affected children). Presence of a genetic etiology was associated with younger age and hemophagocytic lymphohistiocytosis. Ten-year survival for the cohort was 72.4%, and patients with viable genetic diagnoses had a higher survival rate (82%) compared to children without a genetic explanation (48%, P = .03). Survival outcomes for individuals with EBV-associated disease and no genetic explanation were particularly worse than outcomes for subjects with EBV-associated disease and a genetic explanation (17% vs 90%; P = .002). Ascertainment of a molecular diagnosis provided targetable treatment options for up to 18 individuals and led to active management changes for 12 patients. CONCLUSIONS: PLPD defines children at high risk for mortality, and whole exome sequencing informs clinical risks and therapeutic opportunities for this diagnosis.

HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease.

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)-dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.

Physiological C-terminal truncation of α-synuclein potentiates the prion-like formation of pathological inclusions.

α-Synuclein (αsyn) aggregates into toxic fibrils in multiple neurodegenerative diseases where these fibrils form characteristic pathological inclusions such as Lewy bodies (LBs). The mechanisms initiating αsyn aggregation into fibrils are unclear, but ubiquitous post-translational modifications of αsyn present in LBs may play a role. Specific C-terminally (C)-truncated forms of αsyn are present within human pathological inclusions and form under physiological conditions likely in lysosome-associated pathways, but the roles for these C-truncated forms of αsyn in inclusion formation and disease are not well understood. Herein, we characterized the in vitro aggregation properties, amyloid fibril structures, and ability to induce full-length (FL) αsyn aggregation through prion-like mechanisms for eight of the most common physiological C-truncated forms of αsyn (1-115, 1-119, 1-122, 1-124, 1-125, 1-129, 1-133, and 1-135). In vitro, C-truncated αsyn aggregated more readily than FL αsyn and formed fibrils with unique morphologies. The presence of C-truncated αsyn potentiated aggregation of FL αsyn in vitro through co-polymerization. Specific C-truncated forms of αsyn in cells also exacerbated seeded aggregation of αsyn. Furthermore, in primary neuronal cultures, co-polymers of C-truncated and FL αsyn were potent prion-like seeds, but polymers composed solely of the C-truncated protein were not. These experiments indicated that specific physiological C-truncated forms of αsyn have distinct aggregation properties, including the ability to modulate the prion-like aggregation and seeding activity of FL αsyn. Proteolytic formation of these C-truncated species may have an important role in both the initiation of αsyn pathological inclusions and further progression of disease with strain-like properties.

Prehospital Administration of Epinephrine in Pediatric Anaphylaxis - A Statewide Perspective.

OBJECTIVE: Timely administration of epinephrine is critical in the treatment of anaphylaxis. This study sought to determine the frequency of administration of epinephrine by EMS providers caring for pediatric patients in the prehospital setting. METHODS: We examined data from the NC EMS database (PreMIS) from 2010-3 to determine frequency of epinephrine administration in pediatric patients with anaphylaxis. We studied patients <18 years of age with an EMS provider impression of "allergic reaction." Anaphylaxis was present if there was hypotension (defined as SBP < 90 or DBP < 45 for patients age 11 and older, and SBP < 70 + (2 × age) for patients ages 0-10), or impaired respirations (defined as description of labored or absent respirations, or RR < 12 or > 30). We determined the overall frequency of epinephrine administration. A multivariate logistic regression was then constructed to examine the impact of the following variables on appropriate epinephrine administration: age < 10, non-white race, rural county of case origin, duration of transportation from scene, and presence of a paramedic. RESULTS: A total of 504 patients met inclusion criteria, of which 471 demonstrated anaphylaxis as previously defined. A total of 153 patients with anaphylaxis received epinephrine (32.4%, 95% CI 28.3-36.9%). Age < 10 was associated with increased odds of not receiving epinephrine appropriately (OR 2.90, 95% CI 1.85-4.54, p < 0.001). Other variables did not have statistically significant impact on epinephrine administration. CONCLUSION: There are missed opportunities for prehospital administration of epinephrine in pediatric patients with anaphylaxis. Very young children (age < 10) had increased odds for not receiving epinephrine.

Recent trends in gas-phase ammonia and PM2.5 ammonium in the Southeast United States.

UNLABELLED: Ammonia measurements from the Southeastern Aerosol Research and Characterization (SEARCH) study network were analyzed for trends over 9 yr (2004-2012) of observations. Total ammonia concentrations, defined as the sum of gas-phase ammonia and fine particle ammonium, were found to be decreasing by 1-4% yr(-1) and were qualitatively consistent with ammonia emission estimates for the SEARCH states of Alabama, Georgia, Mississippi, and Florida. On the other hand, gas-phase ammonia mixing ratios were found to be slightly rising or steady over the region, leading to the observation that the gas-phase fraction of total ammonia has steadily increased over 2004-2012 as a result of declining emissions of the strong acid precursor species sulfur dioxide (SO2) and nitrogen oxides (NOx) and consequent reduced partitioning of ammonia to the fine particle phase. Because gas-phase ammonia is removed from the atmosphere more rapidly than fine particle ammonium, an increase in the gas-phase fraction of total ammonia may result in shifted deposition patterns as more ammonia is deposited closer to sources rather than transported downwind in fine particles. Additional long-term measurements and modeling studies are needed to determine if similar transitions of total ammonia to the gas phase are occurring outside of the Southeast and to assess if these changes are impacting plants and ecosystems near major ammonia sources. Unusually high ammonia concentrations observed in 2007 in the SEARCH measurements are hypothesized to be linked to emissions from wildfires that were much more prevalent across the Southeast during that year due to elevated temperatures and widespread drought. Although wildfires are currently estimated to be a relatively small fraction (3-10%) of total ammonia emissions in the Southeast, the projected increased incidence of wildfires in this region as a result of global climate change may lead to this source's increased importance over the rest of the 21st century. IMPLICATIONS: Ammonia concentrations from the Southeastern Aerosol Research and Characterization study (SEARCH) network are analyzed over the 9-yr period 2004-2012. Total ammonia (gaseous ammonia+PM2.5 ammonium) concentrations declined at a rate of 1-4% yr(-1), consistent with U.S. Environmental Protection Agency (EPA) emission estimates for the Southeast United States, but the fraction of ammonia in the gas phase has risen steadily (+1-3% yr(-1)) over the time period. Declining emissions of SO2 and NOx resulting from imposed air quality regulations have resulted in decreased atmospheric strong acids and less ammonia partitioning to the particle phase, which may impact the amount and overall pattern of ammonia deposition.

Rituximab therapy in a patient with low grade B-cell lymphoproliferative disease and concomitant acquired angioedema.

Acquired angioedema is often associated with significant morbidity. An underlying lymphatic malignancy, autoimmune disorder, adenocarcinoma, or other malignancy may be present. Screening for these disorders should occur in all patients with acquired angioedema as treatment may result in resolution of angioedema.

Sclerosing encapsulating peritonitis (cocoon bowel) presenting after laparotomy for splenic abscess.

Sclerosing encapsulating peritonitis, previously referred to as cocoon bowel, is a rare cause of intestinal obstruction that often results in obstruction due to the development of a fibrous enhancing membrane that encases multiple small bowel loops. We present a case of a patient who presented to our institution with abdominal distension and guarding. Computed tomography was obtained which revealed findings concerning for sclerosing encapsulating peritonitis. Sonographic imaging was also obtained and provides correlative imaging.

Dendritic filopodia, Ripped Pocket, NOMPC, and NMDARs contribute to the sense of touch in Drosophila larvae.

BACKGROUND: Among the Aristotelian senses, the subcellular and molecular mechanisms involved in the sense of touch are the most poorly understood. RESULTS: We demonstrate that specialized sensory neurons, the class II and class III multidendritic (md) neurons, are gentle touch sensors of Drosophila larvae. Genetic silencing of these cells significantly impairs gentle touch responses, optogenetic activation of these cells triggers behavioral touch-like responses, and optical recordings from these neurons show that they respond to force. The class III neurons possess highly dynamic dendritic protrusions rich in F-actin. Genetic manipulations that alter actin dynamics indicate that the actin-rich protrusions (termed sensory filopodia) on the class III neurons are required for behavioral sensitivity to gentle touch. Through a genome-wide RNAi screen of ion channels, we identified Ripped Pocket (rpk), No Mechanoreceptor Potential C (nompC), and NMDA Receptors 1 and 2 (Nmdars) as playing critical roles in both behavioral responses to touch and in the formation of the actin-rich sensory filopodia. Consistent with this requirement, reporters for rpk and nompC show expression in the class III neurons. A genetic null allele of rpk confirms its critical role in touch responses. CONCLUSIONS: Output from class II and class III md neurons of the Drosophila larvae is necessary and sufficient for eliciting behavioral touch responses. These cells show physiological responses to force. Ion channels in several force-sensing gene families are required for behavioral sensitivity to touch and for the formation of the actin-rich sensory filopodia.

Alternatives to mammalian pain models 2: using Drosophila to identify novel genes involved in nociception.

Identification of the molecules involved in nociception is fundamental to our understanding of pain. Drosophila, with its short generation time, powerful genetics and capacity for rapid, genome-wide mutagenesis, represents an ideal invertebrate model organism to dissect nociception. The fly has already been used to identify factors that are involved in other sensory systems such as vision, chemosensation, and audition. Thus, the tiny fruit fly is a viable alternative to mammalian model organisms. Here we present a brief primer on techniques used in screening for thermal and/or mechanical nociception mutants using Drosophila.

The role of the RING-finger protein Elfless in Drosophila spermatogenesis and apoptosis.

elfless (CG15150, FBgn0032660) maps to polytene region 36DE 5' (left) of reduced ocelli/Pray for Elves (PFE) on chromosome 2L and is predicted to encode a 187 amino acid RING finger E3 ubiquitin ligase that is putatively involved in programmed cell death (PCD, e.g., apoptosis). Several experimental approaches were used to characterize CG15150/elfless and test whether defects in this gene underlie the male sterile phenotype associated with overlapping chromosomal deficiencies of region 36DE. elfless expression is greatly enhanced in the testes and the expression pattern of UAS-elfless-EGFP driven by elfless-Gal4 is restricted to the tail cyst cell nuclei of the testes. Despite this, elfless transgenes failed to rescue the male sterile phenotype in Df/Df flies. Furthermore, null alleles of elfless, generated either by imprecise excision of an upstream P-element or by FLP-FRT deletion between two flanking piggyBac elements, are fertile. In a gain-of-function setting in the eye, we found that elfless genetically interacts with key members of the apoptotic pathway including the initiator caspase Dronc and the ubiquitin conjugating enzyme UbcD1. DIAP1, but not UbcD1, protein levels are increased in heads of flies expressing Elfless-EGFP in the eye, and in testes of flies expressing elfless-Gal4 driven Elfless-EGFP. Based on these findings, we speculate that Elfless may regulate tail cyst cell degradation to provide an advantageous, though not essential, function in the testis.

reduced ocelli encodes the leucine rich repeat protein Pray For Elves in Drosophila melanogaster.

The ocelli are three simple photoreceptors on the vertex of the fruit fly head. We sought to identify the gene encoded by the classical ocellar mutant, reduced ocelli (rdo). Deficiency and inversion breakpoint mapping and P-element induced male recombination analyses were performed and Pray For Elves (PFE; CG15151; Fbgn0032661) emerged as a promising candidate for the rdo phenotype. The PFE locus maps to polytene region 36E on chromosome 2L between elfless (Fbgn0032660) and Arrestin 1 (Fbgn0000120). FlyBase annotation predicts that PFE encodes a serine/threonine kinase, yet protein prediction programs revealed no kinase domain. These analyses suggest that PFE simply encodes a leucine rich repeat molecule of unknown function, but presumably functions in nervous system protein-protein interaction. Two classical spontaneous alleles of rdo, rdo(1) and rdo(2), were characterized and the underlying mutations result from a small deletion spanning exon 1/intron 1 and a B104/roo insertion into the 3'UTR of PFE, respectively. Transposase-mediated excisions of several P-elements inserted into the PFE locus revert the rdo phenotype and a full-length PFE cDNA is sufficient to rescue rdo. A Gal4 enhancer trap reveals a broad adult neural expression pattern for PFE. Our identification and initial characterization of the rdo locus will contribute to the understanding of neurogenesis and neural development in the simple photoreceptors of the Drosophila visual system.

Stimulation of human breast carcinoma cell invasiveness and urokinase plasminogen activator activity by glucose deprivation.

Glucose deprivation has been shown to increase the invasive and metastatic potential of tumour cells. In the present study, we determined whether the enhanced tumour cell invasiveness resulting from glucose deprivation is linked to increased activity of enzymes required for extracellular matrix degradation. Results of in vitro invasion assays revealed that the invasiveness of human MDA-MB-231 and MCF-7 breast carcinoma cells and MCF-10A1 normal breast cells was, respectively, 3.9-, 2.9-, and 2.1-fold higher when they were incubated under glucose-deprivation (0.2 mM glucose) than when incubated under physiological blood glucose levels (5 mM). This effect of glucose deprivation on invasion correlated with increased urokinase plasminogen activator (uPA) and plasmin activity. Glucose deprivation did not increase the levels of gelatinase and plasminogen activator inhibitor-1 secretion, or the expression of cell-associated uPA receptor. To determine whether the increased invasiveness resulting from glucose deprivation is causally linked to increased uPA activity, invasion assays were conducted using MDA-MB-231 cells incubated in 0.2 mM or 5 mM glucose in the presence of a neutralising anti-uPA antibody. Results revealed that the anti-uPA antibody significantly inhibited invasion in a dose-dependent manner and to a much greater extent in cells incubated in 0.2 mM glucose than in cells incubated in 5 mM glucose. These results suggest that low glucose levels in malignant cancers increase tumour cell invasiveness by stimulating uPA and plasmin activity.

Inhibition of human trophoblast invasiveness by high glucose concentrations.

CONTEXT: Trophoblast invasion of the uterus is regulated by local microenvironmental factors. OBJECTIVE: Because certain conditions may affect uterine glucose levels during placentation, the aim of this study was to determine the effect of glucose concentration on trophoblast invasion. RESULTS: Compared with incubation in 0.2 and 2.5 mm glucose, a 24-h incubation in increasing glucose concentrations (5 and 10 mm) resulted in up to a 62% inhibition (P < 0.01) of the in vitro invasiveness of immortalized HTR-8/SVneo trophoblasts. This decreased invasiveness in 5 and 10 mm glucose was paralleled by inhibition of a plasminogen activator (PA) activity corresponding to active urokinase-type PA (uPA). Inhibition of pro-uPA binding to the uPA receptor decreased the invasiveness of cells incubated in 0.2 and 2.5 mm glucose to levels observed in cells incubated in higher glucose concentrations (P < 0.05). Gelatin zymography and Western blot analysis revealed that the levels of matrix metalloproteinase-2 and -9, PA inhibitor-1, and uPA receptor were unaffected by glucose. Glucose transporter-1 levels were 26 and 34% higher in cells cultured in 2.5 and 0.2 mm glucose, respectively, vs. 5 or 10 mm glucose (P < 0.05). In contrast, glucose transporter-3 levels were not affected by incubation in various glucose concentrations. CONCLUSIONS: These findings indicate that high glucose concentrations inhibit the invasiveness of HTR-8/SVneo cells by preventing uPA activation. Therefore, through its effects on uPA activity, glucose may be an important regulator of trophoblast invasiveness during implantation and placentation.

Activated macrophages inhibit human cytotrophoblast invasiveness in vitro.

Pre-eclampsia is associated with inadequate cytotrophoblast invasion and remodeling of the uterine spiral arterioles, as well as by an aberrant maternal immune response. This study determined the effect of activated macrophages and one of its products, tumor necrosis factor (TNF)-alpha, on cytotrophoblast invasiveness. Coculture with human lipopolysaccharide-activated macrophages decreased the ability of immortalized HTR-8/ SVneo human trophoblast cells to invade through reconstituted extracellular matrix (P < 0.05). This effect of activated macrophages on trophoblast invasiveness was paralleled by abrogation of a 55-kDa caseinolytic activity corresponding to prourokinase plasminogen activator (pro-uPA) and an increased secretion of plasminogen activator inhibitor 1 (PAI1), as determined by gel zymography and ELISA, respectively. Coculture with nonactivated macrophages did not significantly affect trophoblast invasiveness or pro-uPA and PAI1 secretion. Activated macrophages secreted detectable levels of TNF, and administration of exogenous TNF significantly decreased trophoblast invasiveness (P < 0.05), increased the secretion of PAI1 (P < 0.01), and completely inhibited the pro-uPA-associated caseinolytic activity by binding to the TNF receptor 1. Moreover, addition of up to 10 ng/ml of TNF did not increase the rate of apoptosis in HTR-8/SVneo cells. Finally, the increased secretion of PAI1 by trophoblast cells cocultured with activated macrophages was significantly inhibited when a neutralizing anti-TNF antibody was added to the cocultures. These results suggest that the aberrant presence of activated macrophages around uterine vessels may contribute to inadequate trophoblast invasion and remodeling of the uterine spiral arterioles. Thus, the presence of activated macrophages may be important in the etiology of pre-eclampsia.

Drosophila N-cadherin mediates an attractive interaction between photoreceptor axons and their targets.

Classical cadherins have been proposed to mediate interactions between pre- and postsynaptic cells that are necessary for synapse formation. We provide the first direct, genetic evidence in favor of this model by examining the role of N-cadherin in controlling the pattern of synaptic connections made by photoreceptor axons in Drosophila. N-cadherin is required in both individual photoreceptors and their target neurons for photoreceptor axon extension. Cell-by-cell reconstruction of wild-type photoreceptor axons extending within mosaic patches of mutant target cells shows that N-cadherin mediates attractive interactions between photoreceptors and their targets. This interaction is not limited to those cells that will become the synaptic partners of photoreceptors. Multiple N-cadherin isoforms are produced, but single isoforms can substitute for endogenous N-cadherin activity. We propose that N-cadherin mediates a homophilic, attractive interaction between photoreceptor growth cones and their targets that precedes synaptic partner choice.